Methyl seq combinatorial dual indexing kit

The cffDNA was extracted from serum using a Maxwell RSC cffDNA Plasma Kit (Promega; AS1480) by the Primate Assay Laboratory Core at the California National Primate Research Center. The brain DNA was isolated from tissue stored in DNA/RNA shield (Zymo Research; R1100-250) using the Quick-DNA Miniprep Plus kit workflow on a Tecan instrument by Zymo Research. Brain DNA was fragmented using a E220 focused-ultrasonicator (Covaris; 500239). DNA was bisulfite converted using the EZ DNA Methylation-Lightning Kit (Zymo Research; D5031). WGBS library preparation was performed via the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences; 30096) with the Methyl-Seq Combinatorial Dual Indexing Kit (Swift Biosciences; 38096) according to the manufacturer’s instructions. The primary cffDNA libraries were prepared by Swift Biosciences and the brain libraries were prepared by the UC Davis Genome Center. The primary cffDNA and brain library pools were sequenced by the UCSF Center for Advanced Technology (CAT) core facility on the Illumina NovaSeq 6000 S4 for 150 bp paired end reads. The pilot cffDNA library pool utilized the Methyl-Seq Set A Indexing Kit (Swift Biosciences; 36024) and was sequenced by the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center on an Illumina HiSeq 4000 for 90 bp single reads.

DNA was isolated from cells using the Qiagen AllPrep DNA/RNA Minikit and quantified using a Qubit fluorometer. Prior to library preparation, sample DNA was spiked with unmethylated lambda phage DNA (Promega) at a concentration of 5 ng lambda DNA/1 μg sample DNA. DNA was fragmented to approximately 350 bp using a Covaris M220 Sonicator, and bisulfite-converted using the Zymo EZ DNA Methylation-Gold Kit according to manufacturer’s instructions (Zymo Research). Bisulfite sequencing libraries were prepared using the Accel-NGS Methyl-Seq DNA Library Kit and Methyl-Seq Combinatorial Dual Indexing Kit (Swift Biosciences). Completed libraries were quantified and QC’ed using Qubit dsDNA HS and Agilent 4200 TapeStation HS DNA1000 assays, respectively.
Sequencing libraries were divided into three pools of six libraries, and WGBS was performed on each pool across three flow cell lanes on an Illumina HiSeq 4000 instrument in 2 × 150PE format using HiSeq 4000 reagents. A PhiX control DNA library was spiked into each lane at 10% of the total to account for the unbalanced base composition inherent in Methyl-Seq libraries. Base calling was done by Illumina Real Time Analysis (RTA) v2.7.7 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.

Nucleic acids were extracted by homogenizing the same half of placenta and brain tissue using a TissueLyser II (Qiagen) followed by the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) according to the manufacturer’s instructions. For the low-pass WGBS libraries, DNA was sonicated to ~350 bp using a E220 focused-ultrasonicator (Covaris) and bisulfite converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer’s instructions. Libraries were prepared via the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) with the Methyl-Seq Combinatorial Dual Indexing Kit (Swift Biosciences) according to the manufacturer’s instructions. The pool of 88 libraries was sequenced on all 4 lanes of an NovaSeq 6000 S4 flow cell (Illumina) for 150 bp paired end reads, which yielded ~65 million unique aligned reads (~6X genome cytosine coverage) for each sample. For the RNA-seq libraires, RNA integrity (RIN > 7) was confirmed using a Bioanalyzer Eukaryotic Total RNA Nano Assay (Agilent). Libraries were prepared with the KAPA mRNA HyperPrep kit (Roche) and NEXTFLEX Unique Dual Index Barcodes (PerkinElmer). The pool of 88 libraries was sequenced on 1 lane of a NovaSeq 6000 S4 flow cell (Illumina) for 150 bp paired end reads, which yielded approximately 25 million uniquely mapped reads for each sample.